i. Field of the Invention
The field of this invention is a process for the production of viruses in microcarrier cell culture. Perfused MRC-5 cells cultured on glass-coated microcarriers and infected with hepatitis A virus exemplifies the process.
ii. Background
Many viruses and therapeutic proteins are produced by anchorage dependent cells where cell attachment to a surface is a prerequisite for cell growth and proper function of the cell line. When relatively small quantities are required, multiple T-flasks and Roller bottles have been traditionally used to supply the required surface area. Other commercially available systems such as NUNC CELL FACTORIES cell culture system and COSTAR CUBES cell culture system have substantially increased surface area and thus increased productivity per bioreactor unit. However, these systems still require multiple bioreactor units for large quantities and are therefore limited in scale-up potential for commercial production.
Various packed bed systems have also been developed, including hollow fiber reactors, packed beds of spheres, packed beds of randomly oriented fibers, and porous ceramic monoliths. These systems have documented difficulties with maintaining nutrient supply to the cells in the reactor due to the reactor configuration and restrictions of nutrient transport paths due to cell growth. Hollow fiber reactors rely on diffusion and Starling flow to supply medium flow across the cell compartment [see J. M. Piret (1989), B.c.D Thesis, Dept. of Chem. Eng., Mass. Inst. Technology, August 1989]. Diffusive nutrient penetration is only adequate if the depth of the cell accumulation on the fibers is controllable and uniform, which it is not in these reactors. Starling flow decreases as cell growth occurs, due to increased flow resistance offered by the cell growth. This reduces mixing within the cell growth chamber. Randomly packed beds of beads or fibers rely on forced convection, and are both prone to channeling through paths of least hydraulic resistance, bypassing the areas where surface area is most dense, and hence likely to contain cells. Porous ceramic monoliths are better in this respect, but these suffer from the second factor, biofouling. As the cells grow on the attachment surface, they can constrict and occlude the paths where medium flow occurs [J. E. Putnam et al., Ann. Mtg. Soc. Ind. Microbiol., Orlando, Fla., Aug. 1, 1990]. In the case of the ceramic monolith, cell growth results in restriction of the channel, such that increased hydraulic resistance to flow is offered. The medium then preferentially flows to other parallel channels, and the result is that the channel with the heaviest cell growth receives the least medium. Due to these heterogeneities in the cellular microenvironment and limited scale-up potential, these reactors have had limited success; no U.S. licensed human vaccines or therapeutics are known to be produced in these systems.
The use of solid static mixer elements in cell and virus culture is known, see Grabner and Paul, U.S. Pat. No. 4,296,204. The use of a mesh for culturing primary tissues comprised of several cell types is found in U.S. Pat. No. 4,963,489 and 5,160,490. The tissue resulting from a mesh culture of stromal fibroblasts is claimed in U.S. Pat. No. 4,963,489. Using motionless mixing elements as the surface for cell growth provides uniform nutrient transport to the cell population. The scale-up, cleaning, and sterilization issues remain a challenge for commercial application of these bioreactor systems. In addition, the biomass in these systems cannot be directly monitored during the cultivation. Therefore, indirect measures of cell mass must be used to characterize the performance of the bioreactor. Due to the same reasons, removal of a cell associated product can also be problematic with these reactor configurations.
Microcarrier technology provides a large amount of surface area for cell growth on small, spherical beads (90 to 250 microns in diameter) which are suspended in a stirred tank bioreactor. High surface to volume ratios can be achieved resulting in a highly efficient production system on a bioreactor volume basis. The technology provides a homogeneous cell culture environment with the capability to quantify cell mass and harvest cell associated product during the cultivation. Since the bioreactor is a stirred tank, well established cleaning and sterilization procedures, as well as overall tank design is readily available from the fermentation industry for commercial applications. The commercial manufacture of Polio Vaccine using microcarriers at the 1000-1500 liter scale illustrates the scale-up potential of this approach. The commercial production of a Rabies vaccine and a Foot and Mouth Disease Vaccine using microcarrier culture further illustrates the proven scale-up potential of this method.
Although many cell lines and viruses have been propagated on microcarriers, many challenges remain in implementation of this technology at commercial scale. Attaining a low shear environment throughout the cultivation and maintaining a viable culture for sustained product formation through extended cultivation periods can be difficult. Selection of the proper microcarrier and culture conditions is often critical in producing the desired product. The propagation of Hepatitis A is a good example of these challenges. Junker, B. et al., ("Evaluation of a Microcarrier Process for Large Scale Cultivation of Attentuated Hepatitis A," Cytotechnology, Vol. 9, 1-3, 1992) described the evaluation of CYTODEX 3 collagen-coated Sephadex Microcarriers microcarriers as the substratum of MRC-5 cells growth and subsequent infection by hepatitis A virus. A major contributor to the low hepatitis A titers from the microcarrier cultures was attributed to the fact that the cells gradually fell off the beads during the infection period. Based on these results, microcarrier technology was reported to be suboptimal for commercial scale production of hepatitis A virus. Because culture of hepatitis A virus in exemplified in the instant patent disclosure, a brief review of the methods used to culture this virus is in order.
In 1973, Feinstone et al., [Science 182, p 1026] identified the etiologic agent of infectious hepatitis, later known as hepatitis A virus, (HAV), using immune electron microscopy. In vitro culture of hepatitis A virus (HAV) was reported by Provost et al [P.S.E.B.M 160, p 213, 1979] according to a process whereby liver from HAV infected marmosets was used as an inoculurn for liver explant culture and fetal rhesus kidney (FRhK6) cell culture [U.S. Pat. No. 4,164,566]. In a later invention, direct inoculation of a HAV, which had not been previously passaged through a subhuman primate, was successfully used to initiate in vitro propagation of HAV [Provost et al., P.S.E.B.M. 167, p 201 (1981); U.S. Pat. No. 5,021,349].
From this work, attenuation of HAV through in vitro culture was demonstrated. In addition, it was demonstrated that upon repeated passage in vitro, HAV cultures became more productive and replication rate accelerated as the virus became adapted to the cultured cells. A further development was the demonstration of protective efficacy of both the live attenuated virus [Provost; et. al., J. Med. Viol. 20, p 165 (1986)] and the formalin inactivated HAV [U.S. Pat. No. 4,164,566; U.S. Pat. No. 5,021,348; Provost et. al., in Viral Hepatitis and Liver Disease, p 83-86, 1988--Alan R. Liss, Inc.]. From the foregoing work, it has become clear that either an inactivated or attenuated, immunogenic HAV are possible vaccine candidates. However, a reproducible, commercial scale process for production of high purity antigen is needed if a safe HAV vaccine is to be commercially available for use in humans.
Various methods have been described to culture HAV for vaccine production. Thus, Provost et al. (U.S. Pat. No. 5,021,348) described a process whereby, in a preferred method, a cell culture of MRC-5 cells was infected with HAV. According to that disclosure, the virus and cells are grown according to conventional methods in monolayer. In U.S. Pat. No. 4,783,407, HAV was grown in Vero cells (a type of primate kidney cell). In U.S. Pat. No. 4,301,209, high titer HAV production in a hollow fiber capillary unit was described. In U.S. Pat. No. 4,412,002 a process whereby HAV was isolated from persistently infected cells was described. In EP 0 302 692, HAV culture in roller bottles was described. In all of these systems, the large scale production of HAV required for a commercial process was not feasible or was severely limited by the amount of surface area available for cell sheets to be established for HAV infection.
In 1984, Widel, et al., published on the propagation of wild type Hepatitis A in a fetal rhesus monkey kidney (Frh-k) cell line grown on CTYODEX 3 microcarriers at 37.degree. C. (Widell, A. etal., "A Microcarrier Cell Culture System for Large Scale Production of Hepatitis A Virus." J. of Virological Methods, Vol 8, 63-71, 1984). There was no mention of cell attrition or microcarrier aggregation using CTYODEX 3 microcarriers as the growth surface for Frh-k cells. Since the same microcarrier system was determined by Junker et al., to be unsuitable for the production of attenuated virus in MRC-5, these culture systems are clearly very different. Therefore, the MRC-5 human diploid cell line, which is preferable over the Frh-k cell line derived from monkeys for the production of a human vaccine, can not be successfully cultivated to produce Hepatitis A vaccine using the process described by Widel due to the tendency of MRC-5 cells to form microcarrier aggregates. Since the methodology described by Widel is limited to FRH-k cells where aggregation and cell attrition were not addressed, no knowledge of how to overcome these problems encountered when using MRC-5 cells could be gained from this work.
Aggregation of cells in microcarrier cell culture is quite common and has been mentioned in the published literature since the mid 1970s. However, few papers have specifically addressed this topic. A few relevant publications are discussed below:
Varani, et al., (1983) compared the growth of MRC-5 diploid cells and two transformed cell lines on glass coated microcarriers and charged DEAE-dextran microcarriers (Varani, J., etal., "Growth of Three Established Cell Lines on Glass Microcarriers." Biotech. and Bioeng., Vol. 25, 1359-1372, 1983). Microcarrier aggregation occurred with all three cell lines on the glass coated microcarriers while only one continuous cell lines on the glass coated microcarriers while only one continuous cell line aggregated with the dextran microcarrier. Scanning electron microscopic (SEM) analysis illustrated a dramatic difference in the way the cells attach to the two surfaces. In the glass microcarrier cultures, the cells attach by long filopodia while for the DEAE dextran microcarriers the attachment occurs across the entire cell edge. This difference in attachment mechanism may be an important factor in the stability we report of the MRC-5/SOLOHILL glass coated system over MRC-5/dextran systems.
Goetghbeur and Hu (1991) demonstrated that cell aggregates could be induced to form with a number of cell lines in the presence of small charged microspheres with a diameter of about 20 microns (microcarriers are typically 90-250 microns; Goetghebeur, S. and W. S. Hu, "Cultivation of Anchorage-Dependent Animal Cells in Microsphere-Induced Aggregate Culture." Appl. Microbial. Biotech., Vol 34, 735-741, 1991). Two transformed cell lines grown in this way were found not to spread, but rather exist as rounded up multilayered populations. A diploid cell line was also grown on these spheres as aggregates, but the cell shape was irregular, see U.S. Pat. No. 5,114,855. Since the spheres used in Hu's work are much smaller than conventional microcarriers, the aggregate formation described in the instant patent disclosure does not fall in the domain covered by Hu's patent. It is unlikely that a reduction in size to the range in Hu's patent would be applicable for the manufacture of Hepatitis A.
Borys and Papoutsakis (1992) investigated ways to inhibit cell aggregate formation with Chinese hamster Ovary K1 cells grown on CYTODEX 3 microcarriers (Borys, M. C., and E. T. Papoutsakis. "Formation of Bridges and Large Cellular Clumps in CHO-cell Microcarrier Cultures: Effects of Agitation, Dimethyl Sulfoxide, and Calf Serum," Cytotechnology, Vol 8, 237-248, 1992). The emphasis of this work was on overgrowth of transformed cell lines during microcarrier culture and did not address the growth of diploid cell lines as aggregates on microcarriers. Increased agitation was found to reduce aggregation and increase cell death due to the breaking of cellular bridges between microcarriers. Our studies, disclosed herein, with MRC-5 and glass coated microcarriers are in agreement with these results. We have found that agitation did reduce the rate of aggregation of MRC-5 cells in the glass coated microcarrier system with an increase in cell death. We also found aggregation to be an irreversible phenomenon under culture conditions.
A significant amount of work has been done to induce continuous cell lines to grow as cell aggregates in suspension culture. Tolbert, et al., (1980) published an early account of this approach and cited a patent on the "adaptation of cell lines to suspension culture" (Tolbert, W. R. etal., "Cell Aggregate Suspension Culture for Large-Scale Production of Biomolecules, " In Vitro, Vol 16(6), 480-490, 1980). Since MRC-5 cells are human diploid, they require cell spreading for biological activity and are therefore not amenable to this approach.
Thus, while reports exists disclosing that MRC-5 cells can be grown on glass microcarriers as cell-microcarrier aggregates and that Hepatitis A can be produced from a CYTODEX 3 microcarrier culture, as noted above, the instability of the MRC-5 cell/CYTODEX 3 microcarrier system has led those skilled in the art to believe that microcarrier culture is not adaptable to efficient HAV production. Disclosed herein is a uniquely stable microcarrier aggregate system which overcomes previous problems in producing Hepatitis A from MRC-5 cells on microcarriers. This disclosure also identifies methodology developed to integrate the microcarrier process into an existing downstream HAV purification system.